Discussion

In this study, the occurrence of mutations in the APC, CTNNB1 and K-ras genes as well as expression of the hMLH1 protein in tumour tissue of 656 sporadic colorectal cancer cases were investigated. The occurrence of mutations in the CTNNB1 gene, which codes for β-catenin, was rare: only five of 464 tumours analysed were found to have a mutation at one of the phosphorylation sites in exon 3. Truncating mutations in APC and activating mutations in K-ras appeared to occur at similar frequencies. Although tumours harbouring both mutations were relatively rare, mutations in APC and K-ras seemed to occur co-dependently. Nine percent of all tumours (58/656) lacked hMLH1 expression, and in these tumours almost no APC or K-ras mutations was detected. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients who harboured a tumour with an APC and/or K-ras mutation.

The selection of patients included in this study was based on the completeness of analyses of both APC and K-ras genes as well as hMLH1 expression and this led to a considerable reduction in the number of cases that could be included in the analyses presented in this study. The largest reduction (72 cases) was due to incompleteness of the analysis of all fragments comprising the APC mutation cluster region. Tumour DNA was derived from formalin-fixed, paraffin-embedded tumour tissue blocks. Depending on the conditions of fixation and storage, the extracted DNA is more or less fragmented, which may have impaired the analysis of mutations in the APC gene more than in the K-ras gene, since the analysis of the latter is based on the amplification of a smaller gene fragment. It should be emphasized that characteristics of patients (age, sex, family history of colorectal cancer) and tumours (sub-localisation, Dukes' stage and differentiation) of the group under study are similar to the 737 patients for whom tumour material was available and to all 819 patients initially recognized within the cohort (data not shown). Moreover, the K-ras and hMLH1data presented here are similar to the data for K-ras and hMLH1 based on the complete groups (737 and 724 cases, respectively) (data not shown).

Mutations in exon 3 of the CTNNB1 gene leading to loss of one of the phosphorylation sites were rare. Strikingly, all five of these mutations occurred in the proximal colon and three of these also had absent hMLH1 expression. This may indicate that these proximal colon tumours, which often also show mismatch repair deficiency, are more likely to harbour CTNNB1 mutations. This was also found in a study of microsatellite instable colorectal tumours [26]. The WAVE screening technique has not been used previously for formalin-fixed, paraffin-embedded tissue, and therefore it seems plausible that samples harbouring a CTNNB1 mutation have escaped detection. However, all 58 hMLH1 deficient samples were analysed by direct sequencing without a prior screening step, and only three of these samples harboured a CTNNB1 mutation, indicating the low frequency of such mutations.

